title: Moving away from the reference genome: evaluating a peptide sequencing tagging approach for single amino acid polymorphism identifications in the genus Populus.
abstract: The genetic diversity across natural populations of the model organism, Populus, is extensive, containing a single nucleotide polymorphism roughly every 200 base pairs. When deviations from the reference genome occur in coding regions, they can impact protein sequences. Rather than relying on a static reference database to profile protein expression, we employed a peptide sequence tagging (PST) approach capable of decoding the plasticity of the Populus proteome. Using shotgun proteomics data from two genotypes of P. trichocarpa, a tag-based approach enabled the detection of 6653 unexpected sequence variants. Through manual validation, our study investigated how the most abundant chemical modification (methionine oxidation) could masquerade as a sequence variant (Ala→Ser) when few site-determining ions existed. In fact, precise localization of an oxidation site for peptides with more than one potential placement was indeterminate for 70% of the MS/MS spectra. We demonstrate that additional fragment ions made available by high energy collisional dissociation enhances the robustness of the peptide sequence tagging approach (81% of oxidation events could be exclusively localized to a methionine). We are confident that augmenting fragmentation processes for a PST approach will further improve the identification of single amino acid polymorphism in Populus and potentially other species as well.
authors.affiliation: Graduate School of Genome Science and Technology, University of Tennessee , Knoxville, Tennessee 37830, USA.
authors.forename: Paul
authors.initials: P
authors.lastname: Abraham
authors.forename: Rachel M
authors.initials: RM
authors.lastname: Adams
authors.forename: Gerald A
authors.initials: GA
authors.lastname: Tuskan
authors.forename: Robert L
authors.initials: RL
authors.lastname: Hettich
mesh_terms: D018745:Genome, Plant; D008715:Methionine / Q000737:chemistry; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D010940:Plant Proteins / Q000032:analysis* / Q000737:chemistry; D011110:Polymorphism, Genetic; D032107:Populus / Q000737:chemistry / Q000235:genetics*; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D013194:Staining and Labeling / Q000379:methods*; D053719:Tandem Mass Spectrometry
chemical_list: D010940:Plant Proteins; D020543:Proteome; D008715:Methionine